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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

• the present disclosure relates generally to approaches for modifying DNA, and more particularly, to improved compositions and methods for CRISPR-based editing that involve modified proteins.
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• the present disclosure provides improved compositions and methods for modifying DNA substrates, such as chromosomes, plasmids and organelle DNA.
• the composition include modified I-F3 proteins for use in CRISPR systems to modify a DNA substrate.
• the modified proteins include TnsC proteins comprising an insertion or substitution of one or more amino acids; TnsA proteins comprising an insertion or substitution of one or more amino acids; TnsB protein comprising an insertion or substitution of one or more amino acids; and a single protein comprising the amino acid sequence of a TnsA protein and the amino acid sequence of a TnsB protein.
• the single protein may comprise a modified TnsA segment, a modified TnsB segment, and/or an insertion of one or more amino acids between the TnsA and TnsB segments.
• Modified Cas8, Cas5, Cas7, and Cas6 proteins are also provided.
• CRISPR systems that include a guide RNA and one or more modified proteins exhibit a higher transposition frequency relative to an I-F3 system comprising the same guide RNA and I-F3 proteins in unmodified form.
• the described compositions and methods may be used to insert a DNA template into a target chromosome or plasmid in a guide RNA-directed manner.
• Polynucleotides encoding one or more of the described proteins, and methods of using the polynucleotides and the proteins for modifying prokaryotic and eukaryotic cells are also provided.
• Cells modified to comprise the modified proteins and polynucleotides are also provided.
• FIG. 1 Analysis of protein tags for the effect on Guide RNA directed transposition with the Tn6900 element from Aero monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA, TnsB, TnsC are encoded as an operon on an expression plasmid (pACYClac)
• Cas8-5, Cas7, and Cas6 are encoded as an operon on an expression plasmid (pBAD322)
• TniQ or tagged derivatives are encoded on an expression plasmid (pBBRara)
• guide RNA is encoded on an expression plasmid (pCDFara).
• TnsA, TnsB, TnsC are encoded as an operon on an expression plasmid (pACYClac)
• TniQ, Cas7, and Cas6 are encoded as an operon on an expression plasmid (pBAD322)
• Cas8-5 or tagged derivatives are encoded on an expression plasmid (pBBRara)
• guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• FIG. 2 Analysis of protein tags for the effect on Guide RNA directed transposition with the Tn6900 element from Aero monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA, TnsB, TnsC are encoded as an operon on an expression plasmid (pACYClac), TniQ, Cas8-5, and Cas6 are encoded as an operon on an expression plasmid (pBAD322), Cas7 or tagged derivatives are encoded on an expression plasmid (pBBRara), and guide RNA is encoded on an expression plasmid (pCDFara).
• TnsA, TnsB, TnsC are encoded as an operon on an expression plasmid (pACYClac), TniQ, Cas8-5, and Cas7 are encoded as an operon on an expression plasmid (pBAD322), Cas6 or tagged derivatives are encoded on an expression plasmid (pBBRara), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• tagged derivatives percent activity is also shown with respect to the untagged protein (Cas7 or Cas6).
• FIG. 3 Analysis of TnsA and TnsB fusions with different protein tags for the effect on guide RNA directed transposition with the Tn6900 element from Aeromonas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsAB fusion, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsAB fusion generated by insertion of two bp between coding regions to shift to a continuous reading frame including both proteins, or tagged derivatives are encoded on an expression plasmid (pBBRlac), TnsC is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition.
• FIG. 4 Analysis of TnsA and TnsB fusion proteins with different protein tags for the effect on Guide RNA directed transposition with the Tn6900 element from Aero monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsAB fusion, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsAB fusion generated by insertion of two bp between coding regions to shift to a continuous reading frame including both proteins, or tagged derivatives with tags inserted between the proteins as indicated, are encoded on an expression plasmid (pBBRlac), TnsC is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• SV40 Nuclear Localization Sequence
• PKKKRKV SEQ ID NO:533
• alternate NLS NLS alt
• PAAKKKKLD SEQ ID NO:539
• 3xHA YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO:541). All tags are separated by a GSG linker indicated by thick black line.
• FIG. 5 Analysis of protein tags on TnsC for the effect on Guide RNA directed transposition with the Tn6900 element from Aero monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA and TnsB are encoded on an expression plasmid (pBBRlac), TnsC or tagged derivative is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• FIG. 6 Analysis of protein tags on TnsC for the effect on Guide RNA directed transposition with the Tn6900 element from Aer monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA and TnsB are encoded on an expression plasmid (pBBRlac), TnsC or tagged derivative is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• Strep WSHPQFEK (SEQ ID NO:543)
• SV40 Nuclear Localization Sequence PKKKRKV (SEQ ID NO:533)
• alternate NLS NLS alt
• PAAKKKKLD PAAKKKKLD
• NP NLS KRPAATKKAGQAKKKK (SEQ ID NO:540). All tags are separated by a GSG linker indicated by thick black line.
• FIG. 7 Analysis of protein tags on TnsC for the effect on Guide RNA directed transposition with the Tn6900 element from Aero monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA and TnsB are encoded on an expression plasmid (pBBRlac), TnsC or tagged derivative is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• FIG. 8 Analysis of protein tags on TnsC for the effect on Guide RNA directed transposition with the Tn6900 element from Aer monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4).
• TnsA and TnsB are encoded on an expression plasmid (pBBRlac), TnsC or tagged derivative is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 are encoded as an operon on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the mean + standard deviation graphed.
• SV40 Nuclear Localization Sequence PKKKRKV (SEQ ID NO:533)
• 3xMyc EQKLISEEDLEQKLISEEDLEQKLISEEDL (SEQ ID NO:534)
• IxMyc EQKLISEEDL (SEQ ID NO:545). All tags are separated by a GSG linker indicated by thick black line.
• FIG. 9 Analysis of internal positions for the FLAG tag on TnsC for the effect on Guide RNA directed transposition with the Tn6900 element from Aeromonas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition was monitored after inducing transposition with the TnsA, TnsB, and TnsC proteins, guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4), testing with and without TniQ-Cascade (Cas8-5, Cas7, and Cas6) proteins.
• a genetic marker on the mobile plasmid was used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition.
• Transposition is monitored after inducing transposition with the TnsA, TnsB, and TnsC derivatives, guide RNA with atypical repeats flanking a spacer matching a site in lacZ (lacZ4), testing with untagged TniQ-Cascade (Cas8- 5, Cas7, and Cas6) proteins.
• TnsA and TnsB are encoded on an expression plasmid (pBBRlac), TnsC wild type ( ) or with tagged derivatives (Alt, SV40, NP or 3xFLAG).
• a genetic marker on the mobile plasmid is used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition.
• Each example was tested three times with the mean + standard deviation graphed.
• FIG 11 Analysis of the effect of combining fusions and tags on Guide RNA directed transposition with the Tn6900 element from Aer monas salmonicida S44 - Transposition was monitored by the mate-out assay.
• Transposition is monitored after inducing transposition with the TnsA, TnsB, TnsC, TniQ, Cas8-5, Cas7, and Cas6 proteins and guide RNA with atypical repeats.
• TnsA and TnsB or tagged fusion protein are encoded on an expression plasmid (pBBRlac), TnsC or tagged derivative is encoded on an expression plasmid (pACYClac), TniQ, Cas8-5, Cas7 and Cas6 (Q-Cascade) are encoded on an expression plasmid (pBAD322), and guide RNA is encoded on an expression plasmid (pCDFara).
• TnsC - TnsC was either wildtype and untagged (No) or with a C-terminal alternate NLS tag (TnsC-Alt NLS, as in Figures 6).
• TnsAB - TnsA and TnsB were either in their wild-type and unfused form (No) or fused with an intervening NLS and 3xHA tag (Tag).
• Q-Cascade - TniQ, Cas8-5, Cas7, and Cas6 was either in the native operon form as found in the original A.
• a genetic marker on the mobile plasmid is used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition.
• Each example was tested three times with the mean + standard deviation graphed.
• FIG 12 Representative type I-F3 CRISPR-Cas transposons analyzed with the TnsAB and TnsC fusion strategy. Each element is listed with an internal tracing number 0-42 and either the strain identifier or Tn#### number.
• Transposon Tn6022 is not a type I-F3 CRISPR-Cas transposon but is from a sister group that was included as an outgroup to make the similarity tree. The similarity tree was constructed with FastTree using the sequence alignments of TnsA, TnsB, TnsC proteins from all elements made with MUSCLE.
• mini element the left and right transposon ends flanking an antibiotic resistance marker for use as a genetic marker
• Transposition is monitored after inducing transposition with TnsA-NLS-3xHA-TnsB fusion and TnsC with Alt NLS inserted at S304 for Tn6900 or corresponding residue in the alignment for other elements.
• Altered TnsC and TnsAB are encoded as a synthetic operon in the TnsC - TnsAB order with an optimized ribosome loading site sequence inserted between on an expression plasmid (pBAD322) under an arabinose inducible promoter.
• a genetic marker on the mobile plasmid is used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition.
• mini element the left and right transposon ends flanking an antibiotic resistance gene for use as a genetic marker
• TnsC and TnsAB of the following elements Tn6900, Tn6677, Tn7005, Tn7011 are encoded as a synthetic operon in the TnsC - TnsAB order with an optimized ribosome loading site sequence inserted between on an expression plasmid (pBAD322) under an arabinose inducible promoter.
• a genetic marker on the mobile plasmid is used to mate the plasmid into a new host where a genetic marker on the transposon is used to determine the percent of the mobile plasmids that were targeted for transposition. Each example was tested three times with the standard deviation shown.
• the transposition proteins were tested alone (noCascade) or in combination with TniQ, Cas8-5, Cas7, and Cas6 expressed in a synthetic operon with reading frames separated by optimized ribosome loading site sequences with wild-type untagged proteins-Q-Cascade and typical/atypical guide RNA combinations were expressed under arabinose control in a pCDF vector.
• the transposition frequency was monitored with the plasmid encoding transposition machinery and with or without the Q- Cascade and typical/atypical guide plasmids. The percentage in bold indicates the frequency of the on-target transposition event (on-target transposition inactivates the lacZ gene giving colonies that are white on media with X-gal indicator instead of blue).
• Figure 16A shows a multiple sequence alignment of 36 full length TnsC protein sequences performed with Clustal Omega (clustalo Version 1.2.4) (Sievers F., Wilm A., Dineen D., Gibson T. J., Karplus K., Li W ., Lopez R., McWilliam EL, Remmert M., Sbding J., Thompson J.D. and Higgins D.G. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7:539; the disclosure of which is incorporated herein by reference) for the sequences listed in the left column of the alignment. A portion of the sequence alignment corresponding to proposed insertion sites within TnsC is shown.
• Clustal Omega clustalo Version 1.2.4
• Organism names and proteins of the disclosure for the TnsC protein sequences are as shown in Table A, which provides sequences for modified Wild type and modified TnsC proteins; Wild-type TnsA, Wild-type TnsB, Modified TnsAB fusion, Wildtype TnsC. Modified TnsC. Wild-type TniQ and Modified TniQ. For each individual aligned sequence the respective number of the first residue in the portion shown appears at the front of the sequence and the number of the last residue in the portion shown appears at the end of the sequence. Alignment adjustments are shown as dashes and added for convenience but do not represent additions, deletions, or gaps in the actual protein sequence.
• Figure 16B shows a multiple sequence alignment of 28 full length TnsC protein sequences performed with Clustal Omega. A portion of the sequence alignment corresponding to proposed insertion sites within TnsC is shown. Nomenclature of the TnsC protein sequences is as shown in Table A. For each individual aligned sequence the respective number of the first residue in the portion shown appears at the front of the sequence and the number of the last residue in the portion shown appears at the end of the sequence. Alignment adjustments are shown as dashes and added for convenience but do not represent additions, deletions, or gaps in the actual protein sequence.
• the disclosure includes all polynucleotide and amino acid sequences described herein. Each RNA sequence includes its DNA equivalent, and each DNA sequence includes its RNA equivalent. Complementary and anti-parallel polynucleotide sequences are included. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included, including but not limited to sequences included by way of sequence alignments. Sequences of from 40.00%-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included.
• the disclosure includes all polynucleotide and all amino acid sequences that are identified herein by way of a database entry. Such sequences are incorporated herein as they exist in the database on the filing date of this application or patent.
• the disclosure includes all steps and reagents such as proteins and nucleic acids, and all combinations of steps reagents, described herein, and as depicted on the accompanying figures.
• the described steps may be performed as described, including but not necessarily sequentially. Any described reagent(s) and step(s) may be excluded from the claims of this disclosure.
• the described reagents, steps, and systems of this disclosure may comprise or consist of any one or combination of said reagents and steps.
• the disclosure also includes all periods of time and all temperatures described herein.
• the disclosure includes the descriptions of PCT application no. PCT/US2020/22964, filed March 16, 2020, published as PCT publication no. WO 2020/186262, and PCT application no. PCT/US21/22582, filed March 16, 2021, published as PCT publication no. WO 2021/188553, the entire disclosures of each of which are incorporated herein by reference.
• the disclosure includes homologous and orthologous proteins that are found in other prokaryotes. Such homologous and orthologous proteins can be modified at positions that can be determined by one skilled in the art based on demonstrations of modifications of proteins as described herein.
• a reference sequence by which homologous, and orthologous proteins (i.e. orthologs), and amino acid positions within such proteins, can be identified is Aeromonas salmonicida strain S44, which may include plasmid pS44-l, and/or the Aeromonas salmonicida strain S44 and its Tn6900 element.
• Representative sources of proteins that can be modified are described herein including but not limited to figures and tables of this disclosure.
• Modified proteins that are encompassed by this disclosure include proteins that can participate in modification of a DNA substrate as further described herein. Proteins that are modified may have at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or at least 99.5% amino acid sequence identity with a sequence described herein by way of a sequence identifier or reference to a database sequence. Percent sequence identity is defined as the percentage of amino acid residues in a particular sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve a maximum percent sequence identity.
• a homologous protein has at least 80% sequence identity to a described sequence. In embodiments, an orthologous protein has 40% to 79% sequence identity to a described sequence. In embodiments, a homologous or orthologous protein is modified at an amino position that corresponds to a specific location of an amino acid sequence that is described herein.
• a component of the described proteins may comprise a linker, a protein tag, a nuclear localization signal, and proteins that comprise any of: insertion of amino acids, replacement of amino acids, and addition of amino acids internally and on the N-terminus, C-terminus, and combinations thereof, thereby providing modified proteins
• the modified proteins comprises one or more I-F3 proteins, which include I-F3 transposon proteins TnsA, TnsB, TnsC, TniQ, and I-F3b Cas proteins Cas8, Cas5, Cas7, and Cas6.
• I-F3 transposon proteins TnsA, TnsB, TnsC, TniQ, and I-F3b Cas proteins Cas8, Cas5, Cas7, and Cas6 are provided in Table A.
• Representative amino acid sequences for wild type and modified Cas8, Cas5, Cas7, and Cas6 are shown in Table B, with Cas8/5 shown as a fusion protein as further described herein.
• the proteins of this disclosure comprise at least one protein that is from, or comprises modification of, one or more organisms that include any I- F3 transposons, including but not necessarily limited to the I-F3a and I-F3b subbranch of the I-F3 elements.
• I-F3 transposons including but not necessarily limited to the I-F3a and I-F3b subbranch of the I-F3 elements.
• I-F3 systems are described herein in the specification and the figures.
• a protein is derived from an organism by, for example, expressing the protein using an expression vector, or an mRNA that is produced by a user of a described system for modifying a DNA template, as further described herein.
• the modified proteins include but are not necessarily limited to TnsC protein, TnsA protein and TnsB protein.
• the modifications may comprise insertions, substitutions, or amino acids that are added to the N-terminus or C-Terminus of the described proteins.
• the disclosure provides modified TnsC proteins that comprise an insertion or a replacement of endogenous amino acids.
• the insertion is internal to the TnsC protein.
• the replacement is a replacement of endogenous internal TnsC amino acids.
• endogenous it is meant that a replacement comprises a replacement of a wild type amino acid sequence.
• internal it is meant an insertion is not located at the C-terminus or N-terminus of the TnsC protein, although the disclosure includes TnsC and other proteins as described herein that have amino acids added to the C-terminus, N-terminus, or both.
• a modification is made at a position that is at the N-terminus or C-terminus of a described protein.
• a modification is at least one amino acid from an N or C terminus of a described protein, or at a position that is 2-400 amino acids from an N terminus or a C-terminus of a described protein.
• a modification is made between amino acids acid 100 and 250 of a described protein.
• a modification is made between amino acids 130-160 of a described protein.
• a modification is made between amino acids 140 and 150 of a described protein.
• a modification is made N-terminal or C-terminal relative to position 100 of a described protein. In embodiments a modification is made N-terminal or to position 100 of a described protein. In embodiments a modification is made C-terminal relative to position 100 of a described protein. In embodiments a modification is made N- terminal or C-terminal relative to position 300 of a described protein. In embodiments an insertion is made at the amino acid immediately after or before amino acid 143, 145, or 146 of a described protein. In embodiments an insertion is made immediately after or immediately before after amino acid 303, 304, or 305 described herein. All of the modifications described above pertain and their amino acid positions apply to each and every protein described herein.
• the disclosure provides a modified TniQ protein.
• the disclosure provides a modified TnsA protein.
• the disclosure provides a modified TnsB protein.
• an engineered fusion protein comprising a wild type or modified TnsA protein and a wild type or modified TnsB protein.
• An engineered fusion protein comprising a wild type TnsA and wild type TnsB protein of this disclosure is a fusion protein comprising TnsA and TnsB proteins that are not fused in an unmodified system, i.e., the TnsA and TnsB proteins are not produced as a single protein by naturally occurring bacteria.
• a TnsA and TnsB fusion protein comprises an insertion of amino acids between the TnsA and TnsB components of the fusion protein.
• the disclosure comprises a modification of a Cas protein, including but not necessarily limited to Cas5, Cas6, Cas7, Cas8, or Cas8-5.
• Cas5 and Cas8 the Cas8 and Cas5 proteins can be found as a fusion protein in some naturally occurring bacteria.
• the fusion protein may be referred to herein as Cas8/5 or Cas8-5.
• the Cas8 segment, the Cas5 segment, or both may be modified as described herein, including but not limited to amino acid additions and substitutions, representative examples of which are provided in Table B.
• the disclosure provides a modified TnsC protein that comprises an insertion in a segment comprising a sequence Xaai-Xaa2-Xaa3 wherein at least one of the amino acids is a Ser and at least one of the amino acids is a Tyr. In an embodiment one of the amino acids is Ser, one of the amino acids is a Tyr, and the third amino acid is any amino acid.
• the disclosure provides a modified TnsC protein with an insertion of amino acids beginning at or approximately at position 144 or 304, or a combination thereof, of a TnsC protein, or at a corresponding position in a homologous or orthologous protein. In embodiments, in an unmodified TnsC protein a Ser is present at position 304.
• the disclosure provides a combination of TnsA, TnsB, and TnsC, wherein at least one of the TnsA, TnsB, or the TnsC comprises an insertion or replacement of internal amino acids, and/or wherein the TnsA, and TnsB components are provided as an engineered fusion protein that optionally comprises an insertion between the TnsA and TnsB components.
• an insertion between a TnsA and TnsB protein is between amino acids 500-700 of the TnsA or TnsB protein.
• a modification comprises an insertion or replacement of one or more amino acids.
• the modification comprises 2-30 amino acids.
• the modification comprises a randomized sequence.
• the modification comprises an introduced protein purification tag, non-limiting examples of which include FLAG-tags, streptavidin, V5 tags, a tag derived from the c-myc gene product (e.g., a myc tag), and the like.
• only one insertion, only one replacement, or only one addition is made.
• more than one insertion, replacement, or addition, or a combination thereof, is made.
• the replacement or insertion comprises linking amino acids that connect a first component to a second component.
• Suitable amino acid linkers may be mainly composed of relatively small, neutral amino acids, such as glycine, serine, and alanine, and can include multiple copies of a sequence enriched in glycine and serine.
• the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more amino acids.
• the modification comprises a nuclear localization sequence (NLS) that functions in trafficking the modified protein to the nucleus of a cell.
• NLS sequence are known in the art and can be adapted for use with the proteins described herein when given the benefit of the present disclosure.
• the NLS comprises an SV40 NLS. In embodiments, the NLS comprises a nucleoplasmin NLS. In embodiments, the NLS comprises the alternate (Alt) sequence. In embodiments, the
• an insertion or replacement comprises any one or combination, of a repeated sequence in the following table, which also includes a representative linker:
• a protein of this disclosure comprises a contiguous sequence that comprises a linker.
• the linker may separate amino acid sequences of two distinct proteins that are joined in a fusion protein, or may be next to or flank a modification.
• One linker, or more than one linker may be used.
• Amino acid linkers may be mainly composed of relatively small, neutral amino acids, such as glycine, serine, and alanine, and can include multiple copies of a sequence enriched in glycine and serine.
• the linker may comprise from 1-100 amino acids, inclusive, and including all numbers and ranges of numbers there between. In specific and non-limiting embodiments, the linker comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids.
• the linker comprises a segment of a protein from K. oxytoca.
• the K. oxytoca linker comprises the sequence KYAQQNSLFICSFP (SEQ ID NO:547).
• One or more of the proteins may be fused together, with or without other proteins.
• Cas8 and Cas5 are present in a single fusion protein.
• TnsA and TnsB are present in a single fusion protein, as further described herein.
• the proteins are fused to one another without linking amino acids.
• linking amino acids can be included.
• a fusion protein comprising TnsA and TnsB proteins also comprises an NLS.
• proteins described herein may be expressed from a coding sequence that includes a ribosomal skipping sequence.
• Ribosomal skipping sequences are known in the art and include, in non-limiting embodiments, the ribosomal skipping peptides T2A, P2A, E2A, and F2A.
• fusion proteins comprising TnsA and TnsB, and modified TnsC proteins, have been consctructed and determined to function for transposition in a standard mate out assay as demonstrated in the accompanying figures.
• a CRISPR system that includes one or more of the described modified proteins exhibits higher transposition frequency than a control value.
• the control value may be a transposition frequency obtained using one or more modifed proteins that comprises a different modification than the one or more modified proteins that exhibit a higher transposition frequency, as illusrated in the accompnaying figures.
• the modified proteins of this disclosure may also exhibit less off-target transposition than a control value.
• the described modified proteins when used in a CRISPR system exhibit a gain-of-activity phenotype that permits transposition without a CRISPR-Cas effector.
• the disclosure facilitates an increase of transposition efficiency relative to a control, such as transposition from a chromosome to a plasmid, or a plasmid to a chromosome, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
• a control such as transposition from a chromosome to a plasmid, or a plasmid to a chromosome, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
• control comprises transposition frequency exhibited by a system that uses unmodified proteins that are encoded by Aeromonas salmonicida strain S44.
• Transposition efficiency can be determined for transposition events where the transposition comprises transposing an element in cis, e.g., transposition from one location in a chromosome to a different location in the same chromosome.
• an increase of transposition efficiency is obtained using a system comprising at least a first modified protein of this disclosure comprising an internal modification, relative to transposition efficiency of a system comprising the same first modified protein but with a different modification, such as an addition of amino acids at its N or C terminus.
• the disclosure provides systems comprising the described modified proteins.
• the systems comprise one or more of the modified proteins, a guide RNA that is targeted to a selected location in a chromosome or plasmid, and a DNA cargo sequence.
• the guide RNA comprises atypical repeats, such guide RNAs being described in PCT application no. PCT/US2021/22582, from which the description of guide RNAs and atypical repeats, and all organisms, and proteins and CRISPR RNAs encoded by the organisms, is incorporated herein by reference.
• the described systems also provide a DNA cargo sequence for use in insertion into a DNA substrate.
• the DNA cargo sequence can include left and right end transposon sequences.
• the transposon left and right end sequences may also be inserted with a DNA cargo.
• the DNA cargo sequence is inserted into a DNA substrate by cooperation of the described proteins and the targeting RNA to produce the DNA editing.
• Those skilled in the art will be able to understand the terms “left” and “right” transposon sequences, and recognize such sequences.
• the one or more I-F3 proteins may be obtained from, and modified, from any of organism that encode I-F3 proteins.
• an I-F3b protein that is used and/or modified according to this disclosure is encoded by the genome of an organism with an attachment site downstream of the ffs gene encoding the signal recognition particle, and those that are downstream of the downstream of the rsm J gene.
• the described modified proteins are obtained, or derived, from type any I-F3 systems, or type I-B Tn7-CRISPR-Cas systems.
• a “functional fragment” means one or more segments of contiguous amino acids of a polypeptide described herein which retain sufficient capability to participate in target RNA programmed insertion of the DNA insertion template.
• a functional fragment may therefore comprise or consist of, for example, a core domain, a catalytic domain, a polynucleotide binding domain, and the like.
• a single domain, or more than one domain, can be present in a functional fragment.
• compositions and methods of this disclosure are functional in a heterologous system.
• “Heterologous” as used herein means a system, e.g., a cell type, in which one or more of the components of the system are not produced without modification of the cells/system.
• a non-limiting embodiment of a heterologous system is any bacteria that is not Aeromonas salmonicida, including but not necessarily limited to Aeromonas salmonicida strain S44.
• a representative and non-limiting heterologous system is any type of E. coli.
• a heterologous system also includes any eukaryotic cell.
• the heterologous cell is a member of any group that does not endogenously use an I-F3b system.
• the presently described systems are used to insert a DNA insertion template to virtually any position in a bacterial genome, any episomal element, or a eukaryotic chromosome, in an orientation dependent fashion, but in certain instances may require a PAM sequence.
• the system is targeted via a targeting RNA to a sequence in a chromosome in a eukaryotic cell, or to a DNA extrachromosomal element in a eukaryotic cell, such as a DNA viral genome.
• the disclosure includes modifying eukaryotic chromosomes, and eukaryotic extrachromosomal elements, such as DNA in any organelle. Accordingly, the type of extrachromosomal elements that can be modified according to the presently described compositions and methods are not particularly limited.
• systems of this disclosure include a DNA cargo for insertion into a eukaryotic chromosome or extrachromosomal element, or in the case of prokaryotes, a chromosome or a plasmid.
• the disclosure provides for insertion of DNA cargo that can be selected by the user of the system.
• the DNA cargo may be provided, for example, as a circular or linear DNA molecule.
• the DNA cargo can be introduced into the cell prior to, concurrently, or after introducing a system of the disclosure into a cell.
• the sequence of the DNA cargo is not particularly limited, other than a requirement for suitable right and left ends that are recognized by proteins of the system.
• the right and left end sequences that are required for recognition are typically from about 90 - 150-bp in length.
• 90-150 bp length comprises multiple 22bp binding sites for the I- F3b TnsB transposase in the element in each of the ends that can be overlapping or spaced.
• the minimum length of the DNA cargo is typically about 700bp, but it is expected that from 700bp to 120kb can be used and inserted.
• the disclosure provides for insertion of a DNA cargo without making a double-stranded break, and without disrupting the existing sequence, except for residual nucleotides at the insertion site, as is known in the art for transposons.
• the insertion of the DNA cargo occurs at a position that is from approximately 47, 48, or 49 nucleotides from a protospacer in the target (e.g., chromosome or plasmid) sequence.
• the DNA insertion template may be devoid of any sequence that can be transcribed, and as such may be transcriptionally inert.
• Such sequences may be used, for example, to alter a regulatory sequence in a genome, e.g., a promoter, enhancer, miRNA binding site, or transcription factor binding site, to result in knockout of an endogenous gene, or to provide an interval in the dsDNA substrate between two loci, and may be used for a variety of purposes, which include but are not limited to treatment of a genetic disease, enhancement of a desired phenotype, study of gene effects, chromatin modeling, enhancer analysis, DNA binding protein analysis, methylation studies, and the like.
• the DNA sequence comprises a sequence that may be transcribed by any RNA polymerase, e.g., a eukaryotic RNA polymerase, e.g., RNA polymerase I, RNA polymerase II, or RNA polymerase III.
• RNA polymerase e.g., a eukaryotic RNA polymerase, e.g., RNA polymerase I, RNA polymerase II, or RNA polymerase III.
• the RNA that is transcribed may or may not encode a protein, or may comprise a segment that encodes a protein and a noncoding sequence that is functional.
• functional RNAs include any catalytic RNA, or an RNA that can participate in an RNAi-mediated process.
• the functional RNA comprises all or a fragment of an siRNA, an shRNA, a tRNA, a spliceosomal RNA, or any type of micro RNA (miRNA), a snoRNA, or the like.
• the RNA that does not code for a protein encodes a long noncoding RNA (IncRN A).
• the functional RNA may comprise a catalytic segment, and thus may be provided as a ribozyme.
• the ribozyme comprises a hammerhead ribozyme, a hairpin ribozyme, or a Hepatitis Delta Virus ribozyme.
• Such agents can be used, for example, to modulate any RNA to which they are targeted.
• the DNA insertion template includes one or more promoters.
• the promoter may be constitutive or inducible.
• the promoter may be operably linked to a sequence that encodes any protein or peptide, or a functional RNA.
• the DNA insertion template comprises one or more splice junctions.
• the insertion template may comprise a GU near a 5’ end of a coding sequence, and a branch site near the 3’ end of the coding sequence.
• the DNA insertion templates results in exon skipping, or it provides a mutually exclusive exon, or it provides an alternative 5’ splice junction as a donor site, or an alternative 3' splice junction as an acceptor site, or a combination thereof.
• the DNA insertion template reduces or eliminates intron retention.
• the DNA insertion template comprises at least one open reading frame, which may be operably linked to a promoter that is included with the DNA insertion template, or the DNA insertion template is linked to an endogenous cell promoter once integrated.
• the open reading frame and thus the protein encoded by it, is not limited.
• the DNA insertion template comprises an open reading frame that encodes a peptide, e.g., a peptide that can be translated and which may be, for example, from several to 50 amino acids in length, whereas longer sequences are considered proteins.
• a protein encoded by the DNA insertion template includes a cellular localization signal, and thus may be transported to any particular cellular compartment.
• the encoded protein comprises a secretion signal.
• the encoded protein comprises a transmembrane domain, and thus may be trafficked to, and anchored in a cell membrane.
• the anchored protein may comprise either or both of an intracellular domain and an extracellular domain, and may accordingly be displayed on the cells surface, and may further participate in, for example, signal transduction, e.g., the protein comprises a surface receptor.
• a protein encoded by the DNA integrate template comprise a nuclear localization signal.
• a protein encoded by the DNA integrate template comprises one or more glycosylation sites.
• the protein encoded by the DNA insertion template comprises at least one antigenic determinant, e.g., an epitope, and thus may be used to produce cells, such as antigen presenting cells, that may display a peptide comprising an epitope on the cell surface via MHC (e.g, HLA) presentation.
• an antigenic determinant e.g., an epitope
• MHC e.g, HLA
• the protein encoded by the DNA insertion template encodes a binding partner, such as an antibody or antigen binding fragment of an antibody.
• the binding partner comprises an intact immunoglobulin, or as fragments of an immunoglobulin including but not necessarily limited to antigen-binding (Fab) fragments, Fab' fragments, (Fab')2 fragments, Fd (N-terminal part of the heavy chain) fragments, Fv fragments (two variable domains), dAb fragments, single domain fragments or single monomeric variable antibody domains, isolated CDR regions, single-chain variable fragment (scFv), and other antibody fragments that retain antigen binding function.
• Fab antigen-binding
• Fab' fragments fragmentse.g., Fab' fragments, (Fab')2 fragments
• Fd N-terminal part of the heavy chain fragments
• Fv fragments two variable domains
• dAb fragments single domain fragments or single monomeric variable antibody domains
• isolated CDR regions single-chain variable fragment (scFv),
• one or more binding partners are encoded by the DNA insertion template and encode all or a component of a Bi-specific T-cell engager (BiTE), a bispecific killer cell engager (BiKE), or a chimeric antigen receptor (CAR), such as for producing chimeric antigen receptor T cells (e.g. CAR T cells).
• the binding partners are multivalent, and as such may include tri-specific antibodies or other tri-specific binding partners.
• the DNA insertion template encodes a T cell receptor, and thus may encode both an alpha and beta chain T cell receptor, or separate DNA insertion template s may be used.
• the DNA insertion template encodes an enzyme; a structural protein; a signaling protein, a regulatory protein; a transport protein; a sensory protein; a motor protein; a defense protein; or a storage protein.
• the DNA insertion template encodes a protein or peptide hormone.
• the DNA insertion template encodes hemoglobin.
• the DNA insertion template encodes all or a segment of dystrophin.
• the DNA insertion template encodes a rod or cone protein.
• the DNA insertion template encodes a selectable or detectable marker.
• the detectable marker comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced GFP (eGFP), mCherry, and the like.
• the DNA insertion template encodes an auxotrophic marker, such as for use in yeast.
• the DNA insertion template encodes one or more proteins that are involved in a metabolic pathway.
• the DNA insertion template encodes a peptide or protein that is intended to stimulate an immune response, which may be a humoral and/or cell mediated immune response, and may also include a peptide or protein that is intended to induce tolerance, such as in the case of an autoimmune disease or an allergy.
• the DNA insertion template encodes a Toll-like-receptor (TLR), or a TLR ligand, which may be an agonist or an antagonistic TLR ligand.
• TLR Toll-like-receptor
• the DNA insertion template comprises a sequence that is intended to disrupt or replace a gene or a segment of a gene.
• the disclosure includes producing both knock in and knock out gene modifications in cells, and transgenic non-human animals that contain such cells, as well as prokaryotic cells modified in a similar manner.
• the transposable DNA cargo sequence is inserted into the chromosome or extrachromosomal element within a 5 nucleotide sequence that includes the nucleotide that is located 47 nucleotides 3’ relative to the 3’ end of the protospacer.
• a DNA cargo insertion comprises an insertion at the center of a 5bp target site duplication (TSD).
• TSD target site duplication
• a suitable guide RNA directs an editing complex to a DNA target comprising a protospacer adjacent motif (PAM) that is cognate to the protospacer, so that precise integration of a DNA cargo can be achieved.
• PAM protospacer adjacent motif
• the PAM comprises or consists of TACC or CC, NC, or CN (where “N” is any nucleotide).
• N is any nucleotide.
• the I-F3b transposon and I-F3b Cas genes can be expressed from any of a wide variety of existing mechanism that can replicate separately in the cell or be integrated into the host cell genome. Alternatively, they could be expressed transiently from an expression system that will not be maintained. In certain embodiments, the proteins themselves could be directly transformed into the host strain to allow their function.
• the disclosure allows for multiple copies of distinct transposon gene cassettes, multiple copies of Cas genes, CRISPR arrays, and multiple distinct cargo coding sequences to be introduced and to modify genetic material in the same cell.
• the disclosure thus includes second, third, fourth, fifth, or more copies of distinct I-F3b transposon genes, I-F3b Cas genes, and distinct cargo coding sequences.
• the delivery vector can be based on any number of plasmid, bacteriophage or another genetic element, when used in prokaryotes.
• the vector can be engineered so it is maintained, or not maintained (using any number of existing plasmid, bacteriophage or other genetic elements). Delivery of these DNA constructions in bacteria can be by conjugation, bacteriophage or any transformation processes that functions in the bacterial host of interest. Modifications of this system may include adapting the expression system to allow expression in eukaryotic or archaeal hosts.
• the disclosure includes use of at least one NLS in one or more proteins, as described herein and illustrated in the figures.
• a system of this disclosure is introduced into eukaryotic cells using, for example, one or more expression vectors, or by direct introduction of ribonucleoproteins (RNPs).
• expression vectors comprise viral vectors.
• a viral expression vector is used.
• Viral expression vectors may be used as naked polynucleotides, or may comprises any of viral particles, including but not limited to defective interfering particles or other replication defective viral constructs, and virus-like particles.
• the expression vector comprises a modified viral polynucleotide, such as from an adenovirus, a herpesvirus, or a retrovirus, such as a lentiviral vector.
• a baculovirus vector may be used.
• any type of a recombinant adeno- associated virus (rAAV) vector may be used.
• a recombinant adeno- associated virus (rAAV) vector may be used.
• rAAV vectors are commercially available, such as from TAKARA BIO® and other commercial vendors, and may be adapted for use with the described systems, given the benefit of the present disclosure.
• plasmid vectors may encode all or some of the well-known rep, cap and adeno-helper components.
• the expression vector is a self- complementary adeno-associated virus (scAAV). Suitable ssAAV vectors are commercially available, such as from CELL BIOLABS, INC.® and can be adapted for use in the presently provided embodiments when given the benefit of this disclosure.
• compositions, methods, vectors, and kits for use in the present approach to DNA editing can include expression and isolation of the proteins required for this process and carrying out some or all of the process in vitro to allow the assembly of novel DNA substrates. These DNA substrates can subsequently be delivered into living host cells or used directly for other procedures.
• the disclosure includes compositions, methods, vectors, and kits for use in the present approach to DNA editing.
• the disclosure provides a system for modifying a genetic target in bacteria and/or eukaryotic cells.
• the system comprises a first set of I-F3b transposon genes tnsA, tnsB, tnsC, one or more I-F3b tniQ, Cas genes cas8f cas5f cas7f and cas6f wherein at least one of the proteins is modified as described herein, and a sequence encoding a guide RNA as described herein that is functional at least with proteins encoded by the I-F3b Cas genes, wherein at least one of the first set of transposon genes, the Cas genes, and/or or the sequence encoding the first guide RNA are present within and/or are encoded by a recombinant polynucleotide.
• transposition frequency can be determined using, for example, a bacteriophage (i.e. viral) vector that cannot replicate or integrate into the bacterial strain used in the assay. Therefore, while the viral vector injects its DNA into the cell, it is lost during cell replication.
• a bacteriophage i.e. viral
• Encoded in the phage DNA is a miniature Tn7 element where the Right and Left ends of the element flank a gene that encodes resistance to an antibiotic, such as Kanamycin (KanR). If the transposon remains on the bacteriophage DNA the cell will still be killed by the antibiotic because the bacteriophage cannot be maintained in that particular strain of bacteria.
• TnsA, TnsB, TnsC and other required I-F3b transposon proteins and nucleotide sequences described herein are added to the cell, transposition will occur because the transposon can move from the bacteriophage DNA into the chromosome (or plasmid) where it will be maintained and allow a colony of bacteria to grow that is antibiotic resistant. Therefore, when the number of infectious bacteriophage particles are in the assay is known, it permits calculation of a frequency of transposition as antibiotic resistant colonies of bacteria per bacteriophage used in the experiment. Thus, in embodiments, using one or a combination of the I-F3b proteins described herein increases transposition frequency. Accordingly, in some embodiments, one or more I-F3b proteins and guide RNA elements as described herein may be used to enhance CRISPR mediated insertion that is accompanied by the transposonbased constructs that are described herein.
• detectable markers and selection elements can be used.
• transposition frequency can be measured, for example, by a change in expression in a reporter gene.
• Any suitable reporter gene can be used, non-limiting examples of which include adaptations of standard enzymatic reactions which produce visually detectable readouts.
• adaptations of P-galactosidase (LacZ) assays are used.
• transposition of an element from one chromosomal location to another, or from a plasmid to a chromosome, or from a chromosome to a plasmid results in a change in expression of a reporter protein, such as LacZ.
• transposition efficiency is determined by measuring the number of cells within a population that experience a transposition event, as determined using any suitable approach, such as by reporter expression, and/or by any other suitable marker and/or selection criteria.
• the disclosure provides for increased transposition, such as within a population of cells, relative to a control.
• the control can be any suitable control, such as a reference value, or any value using a control experiment with proteins that have different modifications.
• the reference value comprises a standardized curve(s), a cutoff or threshold value, and the like.
• transposition efficiency comprises use of a system of this disclosure to transpose all or a segment of DNA from one location to another within the same or separate chromosomes, from a chromosome to a plasmid, or from a plasmid or other DNA cargo to a chromosome. In embodiments, transposition efficiency is greater than a control value obtained or derived from transposition efficiency using the described system.
• the disclosure provides a system for modifying a genetic target in one or more cells, the system comprising a first set of transposon genes tnsA, tnsB, tnsC, and tniQ, Cas genes cas8f cas5f cas7f and cas6f, which encode at least one modified protein as described herein, and wherein at least two of said proteins are within a fusion protein, and a sequence encoding a guide RNA polynucleotide.
• the disclosure provides a method comprising expressing a guide RNA in cells comprising transposon genes tnsA, tnsB, tnsC, wherein the encoded TnsC protein comprises a modification, and wherein and optionally the TnsA and TnsB proteins are present in a described fusion protein, non-limiting examples of which are provided by the Figures.
• expression vectors such as plasmids, are used to produce one or more than one construct and/or component of the system, and any of their cloning steps or intermediates.
• suitable expression vectors known in the art can be adapted to produce components of this disclosure, including vectors that contain any desirable cargo, but in the context of other components described herein, and atypical repeats.
• any protein of this disclosure may be an Aeromonas salmonicida strain S44 protein, or a derivative thereof,
• the disclosure allows for multiple copies of distinct transposon gene cassettes, multiple copies of Cas genes, CRISPR arrays, and multiple distinct cargo coding sequences to be introduced and to modify genetic material in the same cell.
• the disclosure thus includes second, third, fourth, fifth, or more copies of distinct transposon genes, Cas genes, and distinct cargo coding sequences
• the disclosure provides a system for modifying a genetic target in bacteria and/or eukaryotic cells.
• the system comprises a first set of transposon genes tnsA, tnsB, tnsC, and optionally one or more iniQ.
• the Tns proteins that are provided by this disclosure comprise mutations relative to a wild type sequence.
• a “wild type” sequence as used herein means a sequence that preexists in nature without experimentally engineering a change in the sequence.
• a wild type sequence is the sequence of a transposition element, a non-limiting example of which is the sequence of Aeromonas salmonicida strain S44 plasmid pS44-l, which can be accessed via accession no. CP022176 (Version CP022176.1), such as via www.ncbi.nlm.nih.gov/nuccore/CP022176.
• Non-limiting embodiments of amino acid sequences comprising mutations and/or locations of mutations are described herein, and by way of the following amino acid sequences and accession numbers.
• Enlarged, bold and italicized amino acids signify nonlimiting examples of mutations that are encompassed by this disclosure.
• Enlarged sequences are locations where other mutations may be made, and are also included in this disclosure.
• the disclosure includes amino acid insertions, replacements, and additions, to any of these sequences or their naturally occurring counterparts, the sequence of which are known in the art.
• TnsA (A125D) change from Aeromonas salmonicida strain S44 plasmid pS44-l or TnsA(exact from Aeromonas hydrophila strain AFG SD03)
• TnsB (from Aeromonas salmonicida strain S44 plasmid pS44-l) MDKHNGGLFEDEFVIPQPSTSTSPIDAIQAVLPATVDSFPYVLKVEALHRRDYIL WVEKNLAGGWTEKNLTPLLADAALVLPPPTPNWRTLARWRKIYIQHGRKLVSLIPK HQAKGNARSRLPPSDELFFEQAVHRYLVGEQPSIASAFQLYSDSIRIENLGVVENPIK TISYMAFYNRIKKLPAYQVMKSRKGSYIADVEFKAIASHKPPSRIMERVEIDHTPLDL LLLDDDLLVPLGRPSLTLLIDAYSHCVVGFNLNFNQPSYESVRNALLSSISKKDYVKN KYPSIEHEWPCYGKPETLVVDNGVEFWSASLAQSCLELGINIQYNPVRKPWLKPMIE RMFGIINRKLLEPIPGKTF SNIQEKGD YDPQKD AVMRF
• TnsC from Aeromonas salmonicida strain S44 plasmid pS44-l
• the disclosure also includes additional amino acid changes, such as changes in TnsC, which may include gain-of-activity mutations, in canonical Tn7 (e.g., homologous proteins), including but not necessarily limited to
• Tables A and B provide representative examples of unmodified and modified protein sequences that are included within the scope of the disclosure.
• the disclosure includes a kit comprising one or more expression vector(s) that encodes one or more Cas or other enzymes described herein.
• the expression vector in certain approaches includes a cloning site, such as a poly-cloning site, such that any desirable cargo gene(s) can be cloned into the cloning site to be expressed in any target cell into which the system is introduced or already comprises.
• the kit can further comprise one or more containers, printed material providing instructions as to how to use make and/or use the expression vector to produce suitable vectors, and reagents for introducing the expression vector into cells.
• the kits may further comprise one or more bacterial strains for use in producing the components of the system.
• the bacterial strains may be provided in a composition wherein growth of the bacteria is restricted, such as a frozen culture with one or more cryoprotectants, such as glycerol.
• the kit comprises a vector for expression of a guide RNA comprising a user selected spacer.
• the disclosure comprises delivering to cells a DNA cargo via a system of this disclosure.
• the method generally comprises introducing one or more polynucleotides of this disclosure, or a mixture or proteins and polynucleotides encoding the proteins, which may be also provided with RNA polynucleotides, such as the presently described guide RNAs, into one or more bacterial or eukaryotic cells, whereby the Cas and transposon enzymes/proteins are expressed and editing of the chromosome or another DNA target by a combination of the Cas enzymes and the transposon occurs.
• this disclosure is considered to be suitable for targeting eukaryotic cells, and any microorganism that is susceptible to editing by a system as described herein.
• the microorganism comprises bacteria that are resistant to one or more antibiotics, whereby the editing by the present system kills or reduces the growth of the antibiotic-resistant bacteria, and/or the system sensitizes the bacteria to an antibiotic by, for example, use of cargo that targets an antibiotic resistance gene, which may be present on a chromosome or a plasmid.
• the disclosure is thus suitable for targeting bacterial chromosomes or episomal elements, e.g., plasmids.
• a modification of a bacterial chromosome or plasmid causes the bacteria to change from pathogenic to non- pathogenic.
• bacteria are killed.
• one or all of the components of a system described herein can be provided in a pharmaceutical formulation.
• DNA, RNA, proteins, and combinations thereof can be provided in a composition that comprises at least one pharmaceutically acceptable additive.
• the method of this disclosure is used to reduce or eradicate bacterial cells, and may be used to reduce or eradicate persister bacteria and/or dormant viable but non-culturable (VBNC) bacteria from an individual or an inanimate surface, or a food substance.
• VBNC dormant viable but non-culturable
• the disclosure is considered suitable for editing eukaryotic cells.
• eukaryotic cells that are modified by the approaches of this disclosure are totipotent, pluripotent, multipotent, or oligopotent stem cells when the modification is made.
• the cells are neural stem cells.
• the cells are hematopoietic stem cells.
• the cells are leukocytes.
• the leukocytes are of a myeloid or lymphoid lineage.
• the cells are embryonic stem cells, or adult stem cells.
• the cells are epidermal stem cells or epithelial stem cells.
• the cells are cancer cells, or cancer stem cells.
• the cells are differentiated cells when the modification is made.
• the cells are mammalian cells.
• the cells are human, or are non-human animal cells.
• the non-human eukaryotic cells comprise fungal, plant or insect cells.
• the cells are engineered to express a detectable or selectable marker, or a combination thereof.
• the disclosure includes obtaining cells from an individual, modifying the cells ex vivo using a CRISPR system as described herein, and reintroducing the cells or their progeny into the individual for prophylaxis and/or therapy of a condition, disease or disorder, or to treat an injury, trauma or anatomical defect.
• the cells modified ex vivo as described herein are used autologously.
• cells modified according to this disclosure are provided as cell lines.
• the cells are engineered to produce a protein or other compound, and the cells themselves or the protein or compound they produce is used for prophylactic or therapeutic applications.
• the modification introduced into eukaryotic cells according to this disclosure is homozygous or heterozygous.
• the modification comprises a homozygous dominant or homozygous recessive or heterozygous dominant or heterozygous recessive mutation correlated with a phenotype or condition, and is thus useful for modeling such phenotype or condition.
• a modification causes a malignant cell to revert to a non-malignant phenotype.
• the disclosure includes a pharmaceutical formulation comprising one or more components of a system described herein.
• a pharmaceutical formulation comprises one or more pharmaceutically acceptable additives, many of which are known in the art.
• the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for administration to humans.
• the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intraocular injection.
• the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for topical application.
• the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intravenous injection.
• the pharmaceutical compositions comprise and a pharmaceutically acceptable carrier suitable for injection into arteries.
• the pharmaceutical composition is suitable for oral or topical administration. All of the described routes of administration are encompassed by the disclosure.
• expression vectors, proteins, RNPs, polynucleotides, and combinations thereof can be provided as pharmaceutical formulations.
• a pharmaceutical formulation can be prepared by mixing the described components with any suitable pharmaceutical additive, buffer, and the like. Examples of pharmaceutically acceptable carriers, excipients and stabilizers can be found, for example, in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, the disclosure of which is incorporated herein by reference. Further, any of a variety of therapeutic delivery agents can be used, and include but are not limited to nanoparticles, lipid nanoparticle (LNP), exosomes, and the like. In embodiments, a biodegradable material can be used.
• poly(lactide-co-galactide) is a representative biodegradable material.
• any biodegradable material including but not necessarily limited to biodegrable polymers.
• the biodegradable material can comprise poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(beta-amino esters).
• the biodegradable material may be a hydrogel, an alginate, or a collagen.
• the biodegradable material can comprise a polyester a polyamide, or polyethylene glycol (PEG).
• lipid-stabilized micro and nanoparticles can be used.
• compositions of this disclosure are used for treatment of condition or disorder in an individual in need thereof.
• treatment refers to alleviation of one or more symptoms or features associated with the presence of the particular condition or suspected condition being treated. Treatment does not necessarily mean complete cure or remission, nor does it preclude recurrence or relapses. Treatment can be effected over a short term, over a medium term, or can be a long-term treatment, such as, within the context of a maintenance therapy. Treatment can be continuous or intermittent.
• a system of this disclosure is administered to an individual in a therapeutically effective amount.
• a therapeutically effective amount of a composition of this disclosure is used.
• the term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models.
• An animal model can also be used to determine a suitable concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of components to achieve a desired effect, such as a modification in a threshold number of cells. Additional factors which may be taken into account include the particular gene or other genetic element involved, the type of condition, the age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
• a therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and/or reduces the severity of the disease.
• a therapeutically effective amount may also inhibit or prevent the onset of a disease, or a disease relapse.
• cells modified according to this disclosure are administered to an individual in need thereof in a therapeutically effective amount.
• the disclosure comprises providing a treatment to an individual in need thereof by introducing a therapeutically effective amount a composition of this disclosure, or modified cells as described herein to the individual, wherein the cells comprising the DNA insertion treats, alleviates, inhibits, or prevents the formation of one or more conditions, diseases, or disorders.
• the cells are first obtained from the individual, modified according to this disclosure, and transplanted back into the individual.
• allogenic cells can be used.
• the modified eukaryotic cells can be provided in a pharmaceutical formulation, and such formulations are included in the disclosure.
• a described system of this disclosure is introduced into one or more prokaryotic or eukaryotic cells.
• the prokaryotic cells comprise or consist of gram positive, or gram negative bacteria.
• the bacteria may be non-pathogenic, or pathogenic.
• a described system is introduced into prokaryotic cells (e.g., bacterial or archaeal cells) in the context of a host, e.g., a human, animal, or plant host, e.g., the bacteria are a component of a host’s microbiome or are an abnormal component of a microbiome, e.g., a pathogen.
• delivery of a system described herein results in the stable formation of a recombinant microorganism.
• a recombinant microorganism as generated by a system described herein results in the production of an enzyme or metabolite that can alter the health or metabolism of a host, e.g., a human host.
• delivery of a system described herein results in the inactivation of virulence determinants of a microorganism, e.g., antibiotic resistance or toxin production.
• delivery of a system described herein results in killing of the recipient cell. The system may kill some or all of the cells, or render the cells non-pathogenic and/or sensitive to one or more antibiotics.
• the bacteria are used as a component of a food or beverage product, including but not limited to fermented food and beverages, and dairy products.
• such bacteria comprise Lactic acid bacteria.
• selective delivery to a specific type of bacteria is used by way of a bacteriophage or packaged phagemids that can express all or some of the described components, but wherein the bacteriophage exhibits a specific tropism for a particular type of bacteria.
• a delivery vehicle provides only partial specificity towards targeting particular cells, and additional specificity is provided by the choice of DNA sequence being targeted.
• the described systems are introduced into eukaryotic cells.
• eukaryotic cells include but are not necessarily limited to animal cells, fungi such as yeasts, protists, algae, and plant cells.
• the disclosure provides one or more cells, wherein DNA in the cells comprises at least one inserted DNA insertion template.
• the described cells may be any prokaryotic or eukaryotic cells. Accordingly, the disclosure also provides one or more cells that comprise an inserted DNA sequence.
• the eukaryotic cells comprise animal cells, which may comprise mammalian or avian cells, or insect cells.
• the mammalian cells are human or non-human mammalian cells.
• compositions of this disclosure are administered to avian animals, or to a canine, a feline, an equine animal, or to cattle, including but not limited to dairy cattle.
• the cells that are modified by the approaches of this disclosure are totipotent, pluripotent, multipotent, or oligopotent stem cells when the modification is made.
• the cells are neural stem cells.
• the cells are hematopoietic stem cells.
• the cells are leukocytes.
• the leukocytes are of a myeloid or lymphoid lineage.
• the cells are embryonic stem cells, or adult stem cells.
• the cells are epidermal stem cells or epithelial stem cells.
• the cells are cancer cells, or cancer stem cells.
• the cells are differentiated cells when the modification is made.
• the disclosure includes obtaining cells from an individual, modifying the cells ex vivo using a system as described herein, and reintroducing the cells or their progeny into the individual or a immunologically matched individual for prophylaxis and/or therapy of a condition, disease or disorder, or to treat an injury, trauma or anatomical defect.
• the cells modified ex vivo as described herein are autologous cells.
• the cells are provided as cell lines.
• the cells are engineered to produce a protein or other compound, and the cells themselves and/or the protein or compound they produce is used for prophylactic or therapeutic applications.
• eukaryotic cells made according to this disclosure can be used to create transgenic, non-human organisms.
• one or more modified cells according to this disclosure may be used to perform a gene-drive in a population of animals, including but not necessarily limited to insects.
• the one or more cells into which a described system is introduced comprises a plant cell.
• plant cell refers to protoplasts, gamete producing cells, and includes cells which regenerate into whole plants.
• Plant cells include but are not necessarily limited to cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. Plant products made according to the disclosure are included.
• the disclosure provides an article of manufacture, which may comprise a kit.
• the article of manufacture may comprise one or more cloning vectors.
• the one or more cloning vectors may encode any one or combination of proteins and polynucleotides described herein.
• the cloning vectors may be adapted to include, for example, a multiple cloning site (MCS), into which a sequence encoding any protein or polynucleotide, such as any desired targeting RNA, may be introduced.
• MCS multiple cloning site
• An article of manufacture may include one or more sealed containers that contain any of the aforementioned components, and may further comprise packaging and/or printed material. The printed material may provide information on the contents of the article, and may provide instructions or other indication of how the contents of the article may be used.
• the printed material provides an indication of a disease or disorder that is to be treated using the contents of the article.
• polynucleotides when delivered, they may comprise modified polynucleotides or other modifications, such as phosphate backbone modifications, and modified nucleotides, such as nucleotide analogs. Suitable modifications and methods for making nucleic acid analogs are known in the art. Some examples include but are not limited to polynucleotides which comprise modified ribonucleotides or deoxyribonucleotides.
• modified ribonucleotides may comprise methylations and/or substitutions of the 2' position of the ribose moiety with an — O— lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an — O-aryl group having 2-6 carbon atoms, wherein such alkyl or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group.
• modified nucleotides comprise methyl-cytidine and/or pseudo-uridine.
• the nucleotides may be linked by phosphodiester linkages or by a synthetic linkage, i.e., a linkage other than a phosphodiester linkage.
• inter-nucleoside linkages in the polynucleotide agents include, but are not limited to, phosphodiester, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, morpholino, phosphate triester, acetamidate, carboxymethyl ester, or combinations thereof.
• the DNA analog may be a peptide nucleic acid (PNA).

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• 2023
  • 2023-02-09 EP EP23753673.5A patent/EP4476344A4/de active Pending
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